PROJECT SUMMARY Heart disease is the principal cause of death and disability for both men and women in the US accounting for 40% of all annual deaths. A high cholesterol level, especially of low density lipoprotein (LDL), is a well-known risk factor for this disease. The key atherogenic property of LDL particles involves the formation of atherosclerotic plaques. Since the hepatic LDL receptor is the major determinant of plasma LDL-cholesterol levels, a greater understanding of the regulatory mechanisms that control the expression of the LDL receptor is essential. Proprotein convertase subtilisin/kexin-9 (PCSK9) is a well-known indirect regulator of plasma LDL levels by controlling the number of LDL receptor molecules expressed at the plasma membrane. Preliminary studies in our laboratory suggest that alpha-1-antitrypsin (A1AT) directly interacts with PCSK9 in the medium of cells and prevents the formation of PCSK9/LDL receptor complexes in vitro. A1AT also promotes the removal of PCSK9 from the medium of the cells, but the mechanism for this removal is currently unknown. The long-term objective of this research project is to characterize the molecular mechanisms involved in the A1AT-dependent regulation of PCSK9 expression and function. The major impact of this project will be the identification of an endogenous inhibitor of PCSK9 could lead to the development of diagnostic tests to identify patients and provide them with the best and safest treatment options. Also, this inhibitor could become an alternative treatment for patients that develop intolerance to currently available anticholesterolemic drugs. Based on this, we hypothesized that the levels of LDL receptor protein that is available to bind and remove LDL from the circulation are conditioned to the ratio between A1AT and PCSK9. In this research proposal, we will test this hypothesis through the following specific aims: Aim I: To find common regulators of A1AT and PCSK9 expression; Aim II: To detect critical protein regions in A1AT that influences PCSK9 expression/function; Aim III: To identify the pathway(s) involved in the internalization of PCSK9/A1AT complexes.